Sensorineural hearing loss (SNHL) affects a large proportion of the population, generating significant social and health care costs. Many forms of SNHL feature damage to or loss of cochlear sensory hair cells (HCs), which do not regenerate in mammals. Strategies for hearing restoration are informed by studies of birds and fish, which, unlike mammals, spontaneously regenerate HCs from residual supporting cells (SCs). Notch signaling inhibition has emerged as a promising means of regenerating HCs. In mouse models, however, this approach is inefficient after embryonic stages, suggesting that manipulating additional developmental signals may be required. The FGF signaling system is a promising candidate because tight regulation of FGF signaling is critical to all stages of inner ear development, including HC and SC differentiation. We showed previously that mice with an FGFR3-activating mutation modeling Muenke syndrome, have dominant hearing loss associated with a SC fate switch of two Deiters' cells (DCs) to two pillar cells (PCs). The cell fate switch occurs perinatally and is associated with an expansion of FGF/RAS/MAPK signaling into the prospective DC region. Unexpectedly, hearing and SC fate are restored in these Fgfr3 mutants following genetic reduction of FGF10, a ligand that does not normally activate FGFR3. Remarkably, the SC fate switch still occurs in these rescued animals, but is resolved over time. This is associated with restoration of normal patterns of FGF signaling and shows that seemingly fully differentiated cochlear SCs can reversibly switch fates in an FGF-regulated manner. Although genetic data clearly implicate FGF8 as a ligand for FGFR3 in normal PC differentiation, the SC phenotype of Fgf8 otic conditional knockout mice is weaker than that of the Fgfr3 null mice, suggesting that additional Fgfs are involved. Furthermore, the rescue of Muenke syndrome model phenotypes by Fgf10 heterozygosity begs the question of the normal role of cochlear Fgf10 in the perinatal period. Fgf3 is also expressed near developing SCs, but its role in their development is unknown. These data collectively lead to the hypothesis that FGF10 signals are required for development of Fgfr3P244R/+ phenotypes and that Fgf10 and/or Fgf3 are required together with Fgf8 for normal PC differentiation. This will be tested by temporal and spatial regulation FGF signaling in Muenke syndrome model and wild type mice (Aim 1). Our finding of FGF-regulated supporting cell plasticity and the observations by others that DCs express Fgfr3 into adulthood and that Notch inhibition can promote HC regeneration from SC progenitors, suggest the hypothesis that normal perinatal DCs can be transformed into PCs by forced activation of the RAS/MAPK pathway and that Notch inhibition induced in the context of FGF/RAS/MAPK activation will promote HC differentiation. This will be tested using spatial and temporal modulation of the two signaling pathways in vivo (Aim 2). Completion of the Aims will impact the development of strategies that employ developmental signals for hearing restoration.